A method for discovery of conformation-specific immunotherapy targets

Abstract

The present disclosure provides an improved method for identify proteins having a conformation that is specific to a cell type.

Description

PATENT Attorney Docket No.081906-1492705-244810PC Client Ref. No. SF2022-013 A METHOD FOR DISCOVERY OF CONFORMATION-SPECIFIC IMMUNOTHERAPY TARGETS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No. 63 / 633,573, filed April 12, 2024, which is incorporated by reference for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under R21 CA263299 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] It has recently been shown that certain cell surface antigens can adopt alternative protein conformations in particular types of blood cancers, which can be exploited immunotherapy. Work performed by Hosen et al., Nat Med.2017 Dec;23(12):1436-1443) using hybridoma screening identified an antibody that specifically recognizes multiple myeloma (MM), and later determined that this antibody binds to the activated conformation of the protein integrin ß7, which is only expressed in MM. This led to the speculation that such conformation-specific antigens may exist widely; although, no technology yet existed to find them. A recently developed strategy called “structural surfaceomics”, integrating crosslinking mass spectrometry (XL-MS) with glycoprotein cell surface capture (CSC), also identified an acute myeloid leukemia (AML)-specific confirmation of integrin ß2 as a promising immunotherapy target (Mandal et al., Nature Cancer 4:1592-1609, 2023).

[0004] The method employed to identify the AML-specific integrin ß2 employed a combination of the two existing methods in mass spectrometry-based proteomics, i.e., XL- MS and CSC, as well as the integration of fractionation methods for advanced separation resolution of these highly complex samples. On its own, XL-MS is a powerful method to profile protein structure on a global, proteome-wide scale. XL-MS employs chemical crosslinker small molecules, which contain homobifunctional amine reactive groups connected by a linker that form stable bonds between two Lysine groups on a protein that are 79562690V.1within close proximity of each other. Glycoprotein cell surface capture was then integrated with this technique. Glycoprotein cell surface capture is the predominant technique for cell surface protein characterization via biotin-based chemical labeling and is carried out by chemical oxidation of surface protein glycans (see, e.g., Methods Molecular Biology; Springer Protocols 2012, 909, 1–16). After both biotinylating and crosslinking these cell surface proteins, streptavidin beads are employed to obtain biotin-labeled samples, which can be digested with tryspin, followed by 2-D separation integrating orthogonal chromatographic techniques with phospho-enrichable crosslinkers, namely immobilized metal affinity chromatography (IMAC) and high pH reverse-phase (HpH) chromatography. Eluted peptides were characterized by mass spectrometry and use software algorithms. BRIEF SUMMARY OF THE INVENTION

[0005] The present disclosure provides an improved structural surfaceomics technology that provides an enhanced ability to identify peptides that have a non-canonical protein structure that are specific to a cell population of interest. The methods of the present disclosure employ improved fractionation methods and analytical strategy. In some instances, the methods can identify >1,000 more peptides than what was achieved by previous research in this field (Analytical Chemistry 202294 (10), 4236-4242).

[0006] Thus, in one aspect, the present disclosure provides a method of identifying a protein having a noncanonical structure, compared to a canonical protein database structure that is present on the surface of a population of cells of interest, the method comprising: (a) cross-linking cell surface components comprising lysine that are expressed on the surface of cancer cells present in a population comprising cancer cells, wherein cross-linking comprises contacting the population of cancer cells with a PhoX cross-linking agent; (b) oxidizing and biotinylating cell surface glycans; (c) capturing biotinylated protein and incubating captured proteins with a protease preparation to obtain a preparation comprising cross-linked peptides; (d) fractionating the cross-linked peptide-containing solution to enrich for cross-linked proteins wherein fractionating comprises incubating the preparation of (c) with an immobilized metal affinity agent linked to a solid support; (e) eluting peptide with a charge disrupting solution having a pH of at least 9.0 or higher and processing for liquid chromatography-mass spectroscopy; (f) subjecting a sample prepared from step (e) to trapped ion mobility spectroscopy to identify proteins represented by cross-linked peptides; and 79562690V.1determining the three dimensional structure compared to control protein structure to select a protein having an altered structure compared to the control protein. In some embodiments, the protease preparation comprises trypsin and LysC. In some embodiments, the metal affinity column comprises Fe. In other instances, a processing of step (e) comprises basic reverse phase chromatography using a stepwise acetonitrile gradient. In some embodiments, a population of cells of interest comprises cancer cells, such as from a hematological malignancy. In some embodiments, the hematological malignancy is a myeloid lineage malignancy, such as multiple myelomas or acute myeloid leukemia (AML). BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG.1 Structural surfaceomics profiling of AML identifies tumor-specific conformation of ITGB2. A. Crosslinks not meeting distance constraint (blue) suggest open, active conformation of ITGB2. B. Active conformation of ITGB2 is present on AML cell lines but not B-ALL, which only have inactive ITGB2. C. AML patient blasts express active conformation.

[0008] FIG.2: Mass spectrometry strategy of structural surfaceomics technology, consisting of chemical crosslinking with mass spectrometry (XL-MS) and glycoprotein cell surface capture (CSC). After enrichment, different downstream chromatographic / fractionation methods are used to maximize crosslinked peptide coverage and signal in the mass spectrometer. Adapted from Mandal et al.2023.

[0009] FIG.3. Output of structural surfaceomics experiments from Mandal et al.2023 comparing method employing DSSO cross-linker to improved method employing Phox cross-linker. Number of cross-linked spectral matches detected for first iteration (left bar of each set) and second iteration (right bar of each set) for DSSO and Phox cross-linkers. DETAILED DESCRIPTION OF THE INVENTION

[0010] The terms “a”, “an” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a”, “an” or “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0011] The present disclosure provide an improved method of identifying non-canonical conformations of surface polypeptides present on a cell population of interest that do not conform to a canonical structure. In the context of the present disclosure, “canonical 79562690V.1structure” refers to a structure of a protein as it occurs in a cell from a normal control, as published in the Protein Data Bank (PDB) or predicted from a structural software such as AlphaFold. A “non-canonical structure” refers a protein that comprises a peptide region identified in a cross-linked peptide analyzed in accordance with the methods of the present disclosure where the cross-linked peptide deviates from the lysine Ca-Ca distance (where the alpha carbon is the unique carbon atom in the Lysine side chain) in a known canonical reference structure. Reference to “resting state” refers to a canonical structure. Cross-linked peptides

[0012] The method comprises cross-linking proteins on the surface of cells using a cross- linking agent that comprises a moiety that can , e.g., a trifunctional cross-linking agent such as Disuccinimidyl Phenyl Phosphonic Acid (DSPP), also referred to as “PhoX”; oxidizing and biotinylating cell surface glycans; capture of biotinylated complexes using a biotin- binding moiety, e.g., streptavidin, linked to a solid support; cleaving captured proteins using one or more proteases to generate cross-linked peptides; collecting the cross-linked peptides; enriching cross-linked peptides using metal affinity chromatography; eluting cross-linked peptides with a high pH, e.g., at least about 9.5 or greater, solution, e.g., ammonium hydroxide; performing high pH, e.g., at least about 9.5 or greater, processing eluate for performing liquid chromatography–mass spectrometry (LC-MS), e.g., using trapped ion mobility spectroscopy; identifying proteins represented by the cross-linked peptides; analyzing structure of cross-linked peptides compared to a canonical structure and selecting proteins have a non-canonical structure. The methods is further detailed below.

[0013] Cross-linking can be performed using a number of cross-linking agents that contain metal such that IMAC can be employed for enriching for cross-linked peptide. In some embodiments, the cross-linking agent is PhoX, a trifunctional cross-linking agent comprising a metal and a stable phosphonic acid handle (e.g., Steigenberger et al., ACS Cent. Sci.2019, 5, 9, 1514–1522). Alternative phosphonate-containing cross-linking agents that can be employed are provided in WO2020060412.

[0014] A population of cells of interest is exposed to the cross-linker, which cross-links lysine residues that are in proximity. In some embodiments, the cell population comprises at least 5 x 108or 109cells, and often more. Cell surface glycans are then oxidized and biotinylated to provide biotinylated complexes comprising cross-linked proteins (Wollscheid et al., Nat Biotechnol 27:378–386, 2009). Cross-linked complexes can then be captured 79562690V.1using a biotin binding agent such as streptavidin, or any other biotin binding agent, immobilized to a solid substrate. In typical embodiments, the solid substrate is a bead. Captured complexes are then incubated with one or more proteases that cleave at lysine residues, commonly trypsin and / or lysC. Cross-linked peptides are then eluted from desalting columns to provide a preparation comprising cross-linked peptides. In some embodiments, elution is performed with 80% acetonitrile plus 0.1% TFA.

[0015] The peptide preparation can be enriched for cross-linked peptides using immobilized metal affinity chromatography (IMAC) due to the presence of the phosphonate moiety. Any metal affinity reagent can be used, including, those that employ Ni2+, Fe3+, Ga3+, Al3+, and other. In some embodiments the affinity agent comprises Fe3+. In some embodiments, the affinity agent is Fe3+-NTA.

[0016] Following incubation of the peptide preparation comprising cross-linked peptides with the metal affinity agent, the peptide preparation is eluted from the column using a high pH elution buffer. In some embodiments, the high pH elution buffer has a pH of about 10 or higher. As used in in the present disclosure in the context of pH, “about” refers to a variation of 1% from the stated pH. In some embodiments, ammonium hydroxide is used as the elution buffer.

[0017] Following elution, the eluate is then prepared for liquid chromatography with tandem mass spectrometry (LC-MS / MS) analysis, which may also be referred to herein and LC-MS. In some embodiments, In some embodiments, preparation comprises performing high pH basic reverse phase chromatograph using a stepwise gradient of increasing amounts of acetonitrile, a stepwise gradient employing acetonitrile levels from 6% to 80% that are increased in steps. In some embodiments, the levels of acetonitrile used in the step gradient comprise 6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, 35%, 50%, and 80%.

[0018] Sample are then analyzed by LC / MS-MS. In some embodiments, trapped ion mobility spectroscopy is employed, which provide an additional layer of ion separation that can separate cross-linked peptides by their shape.

[0019] Cross-linked peptides can be identified by determining the proteins that corresponding to the cross-linked peptides using numerous software packages, e.g., (Chen, et al., Nat Commun 10, 3404 (2019)). Peptides can then structurally analyzed to assign astructure. Distance variations in the lysine C -C (typically measure in Angstroms)observed in a cross-linked peptide from the canonical structure of a protein to which the 79562690V.1cross-linked peptide is assigned can be used to determine non-canonical structures that deviate from the reference structure, e.g., using a distance threshold. Cells

[0020] The methods of the present disclosure can be employed to evaluate surfaces of any cell for the presence of non-canonical protein structures. In some embodiments, the cells are cancer cells, e.g., from a hematological cancer. In some embodiments, the cells are neoplastic or dysplastic cells of a myeloid lineage. In some embodiments, the cells are immune cells, such as those that cause autoimmunity or other immune diseases. In some embodiments, the cells are senescent cells. In some embodiments, the cells are infected with an infectious agent such as a bacteria, virus, or parasite. In some embodiments, the cells are neurons or other cells of the nervous system. In some embodiments, the cells are other diseased cells or tissues.

[0021] In some embodiments, the cells are neoplastic or dysplastic cells of a myeloid lineage. Such cells, include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, a chronic myeloproliferative disorder, a myelodysplastic syndrome, adult acute myeloproliferative disorder, or multiple myeloma.

[0022] In other embodiments, the hematological cancer is a leukemia of non-myeloid lineage or lymphoma. For example, in some embodiments, the cancer is Burkitt's lymphoma, central nervous system lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, cutaneous T-cell lymphoma, Hodgkin lymphoma, or non- Hodgkin lymphoma.

[0023] Non-hematological cancers can also be evaluated. Non-limiting examples of cancers include breast cancer, prostate cancer, testicular cancer, renal cell cancer, bladder cancer, liver cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, colorectal cancer, anal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, kidney cancer, head and neck cancer, glioblastoma, mesothelioma, melanoma, chondrosarcoma, or bone or soft tissue sarcoma. Technical section Methodology background 79562690V.1

[0024] Surface proteomics, which integrates crosslinking mass spectrometry (XL-MS) with glycoprotein cell surface capture (CSC), was recently developed to identify an acute myeloid leukemia-specific confirmation of integrin ß2 as a promising immunotherapy target (Mandal et al., Nature Cancer 4:1592-1609, 2023). The present disclosure provides an improved surface proteomics method that provides a pronounced increase in yield of cross- linked peptides, and further demonstrates that this method can be used to identify new targets in cancer cells other than acute myeloid leukemia.

[0025] The following is an illustrative protocol of a method of the present disclosure. The following example employs multiple myelomas cells.

[0026] Myeloma cells (AMO1, MM1.S) were harvested in three batches pro a total of 2.4 billion. After spin washing cells with PBS three times, cell surfaces were crosslinked with 10 mM PhoX. Cell surface glycans were then oxidized (1.6 mM sodium metaperiodate) followed by biotinylation (1 mM biocytin hydrazide).

[0027] After lysis, proteins were captured on streptavidin beads, with a protease mixture comprising trypsin and LysC, and then eluted from desalting columns with 80% ACN + 0.1% TFA. X-linked peptides were enriched using immobilized metal (Fe3+-NTA) affinity chromatography (IMAC) in 80% ACN + 0.1% TFA resuspension / wash buffer with 30%NH4OH elution buffer, followed by high pH reverse phase chromatography before LC-MS / MS analysis. After IMAC, the vacuum dried peptides were resuspended in ammonia water (pH 10) to load onto a packed 5 mg C18 micro-column (3 micrometers, Durashell, Phenomenex) in a 200-microliter pipette tip. The peptides were eluted with 190 microliters of ammonia water with a stepwise gradient of increasing levels of acetonitrile (6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, 35%, 50%, and 80%). The 25%, 30%, 35%, and 50% were combined with the 6%, 9%, 12% and 21% .

[0028] LC / MS-MS data was collected in DDA mode on a timsTOF Pro mass spectrometer with a 60 min LC gradient of 11-26% ACN range before a quick ramp up to 100%.

[0029] Phox-crosslinked peptides were mapped to proteins from a myeloma surfaceome database using the XL-MS analysis software pLink2 and proteins mapped to AlphaFold structures with AlphaCross-XL and distance thresholds analyzed. Using this method we detected over 1500 cell surface crosslinked peptides in multiple myeloma cell lines, which is a pronounounced improvement compared to reported results that yielded fewer than 700 crosslinks (Kamal et al, 2023, supra). 79562690V.1

[0030] We then identified crosslink patterns indicative of a non-canonical protein conformation on tumors not present in the resting protein conformation. AlphaCross-XL was employed to rapidly visualize crosslinks per identified protein and classify distance-violating crosslinks based on a user-defined length threshold. Of the canonical cell surface targets in multiple myeloma models detected, CD38 and CD48 were selected for evaluation because they have been characterized as immunotherapy targets, but with only partial tumor specificity. While detected crosslinks suggest that CD38 adopts the AlphaFold-predicted structure, CD48 contained 10 total crosslinks, of which 7 demonstrated a distance violation. These findings indicate a possible tumor-specific conformation of CD48 that could be therapeutically targeted.

[0031] Initial studies to expand this methodology to solid tumors, such as prostate cancer, have also been undertaken. Initial studies detected 300 cross-linked in prostate tumor models, which can be more challenging due to limited access to the cell surface.

[0032] All patents, patent applications, and other publications cited in this application are incorporated by reference with respect to the material for which they are cited. 79562690V.1

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a protein having a noncanonical structure, compared to a canonical protein database structure that is present on the surface of a population of cells of interest, the method comprising: (a) cross-linking cell surface components comprising lysine that are expressed on the surface of cancer cells present in a population comprising cancer cells, wherein cross- linking comprises contacting the population of cancer cells with a PhoX cross-linking agent; (b) oxidizing and biotinylating cell surface glycans; (c) capturing biotinylated protein and incubating captured proteins with a protease preparation to obtain a preparation comprising cross-linked peptides; (d) fractionating the cross-linked peptide-containing solution to enrich for cross-linked proteins wherein fractionating comprises incubating the preparation of (c) with an immobilized metal affinity agent linked to a solid support; (e) eluting peptide with a charge disrupting solution having a pH of at least 9.0 or higher and processing for liquid chromatography-mass spectroscopy; (f) subjecting a sample prepared from step (e) to trapped ion mobility spectroscopy to identify proteins represented by cross-linked peptides; and determining the three dimensional structure compared to control protein structure to select a protein having an altered structure compared to the control protein.

2. The method of claim 1, wherein the protease preparation comprises trypsin and LysC.The method of claim 1 or 2, wherein the metal affinity column comprises Fe.

4. The method of any one of claims 1-3, processing of step (e) comprises basic reverse phase chromatography using a stepwise acetonitrile gradient. 79562690V.

15. The method of any one of claims 1-4, wherein the population of cells of interest comprises cancer cells.

6. The method of claim 5, wherein the cancer cells are from a hematological malignancy.

7. The method of claim 6, wherein the hematological malignancy is a myeloid lineage malignancy.

8. The method of claim 6, wherein the hematological malignancy is multiple myeloma.

9. The method of claim 6, wherein the hematological malignancy is acute myeloid leukemia. 79562690V.1

Images